Takara primescript first strand cdna synthesis kit

Total RNA was extracted from above-mentioned samples using the Tiangen RNAprep plant kit (Tiangen). We used RNase-free DNase1 to remove genomic DNA contamination before dissolving RNA (Tiangen). Additional PCR reactions and agarose gel electrophoresis were used to recheck the purity of RNA. Then, we used the Takara PrimeScript First-Strand cDNA Synthesis kit (Takara) to synthesize first-strand cDNA. All first-strand cDNA samples were diluted 5 times and stored at −20 °C for real-time quantitative PCR (qRT-PCR) experiments. All specific quantitative primers were designed by Primer Premier 5.0 and are shown in Table S2. Real-time quantitative reverse transcription PCR was performed using the TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) kit (Takara) with the QuantStudio™ 7 Flex Real-Time PCR instrument (Applied Biosystems). A 20-µL reaction system was used, and each reaction contained 0.4 µL gene-specific primers, 0.4 µL ROX Reference Dye, 2 µL cDNA sample, 6.8 µL ddH₂O and 10 µL TB Green Premix Ex Taq reagent. The relative expression level was evaluated based on the 2^−ΔΔCT method, and tonoplast intrinsic protein 41 (TIP41) gene was used as the reference gene [73 (link)].

Total RNA was extracted from the SH-SY5Y cells using easy-spin™ Total RNA Extraction kits (Intron Biotechnology, Seoul, Korea). cDNA synthesis was carried out following the instructions provided with the Takara PrimeScript^® First Strand cDNA Synthesis kit (Takara Bio, Inc., Shiga, Japan). For RT-qPCR, 1 μl of gene primer with SYBR^®-Green (Bio-Rad Laboratories, Hercules, CA, USA) in 20 μl of reaction volume was applied. The sequences of the primers used for the qPCR were as follows: HIF-1α forward, 5′-AGAAACCAC CTATGACCTGC-3′ and reverse, 5′-GTCGTGCTGAATAAT ACCACTC-3′; PHD2 forward, 5′-CAAGGACATCCGAGG CGATAAG-3′ and reverse, 5′-CCGTTACAGTGGCGTATC AGG-3′; and β-actin (as an internal control) forward, 5′-GCAA GCAGGAGTATGACGAG3′ and reverse, 5′-CAAATAAA GCCG CCAATC-3′. All reactions with iTaq™ SYBR-Green Supermix (Bio-Rad Laboratories) were performed using the CFX96™ real-time PCR detection system (from Bio-Rad Laboratories).

Total RNA was extracted from tissue samples using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according the manufacturer’s protocol. RNA was retrotranscribed to cDNA according to the manufacturer’s protocol using the Takara PrimeScript First Strand cDNA Synthesis kit (Takara Bio, Inc.). RT-qPCR analysis was performed using an Agilent MX 3005P system with a SYBR Green PCR Amplification kit (Takara Bio, Inc.). Each qPCR analysis was performed in triplicate. β-actin served as control. The primers used were as follows: CRT sense,5’-GGAGCAGTTTCTGGACGGAG-3’ and anti-sense 5’-ACCGTAGAACTTGCCGGAAC-3’; andβ-actin sense5’-CCTGGCACCCAGCACAAT-3’ and anti-sense 5’-GGGCCGGACTCGTCATAC-3’.